Syndromes Induced by Human Papilloma Virus
Every year approximately half a million new cervical cancer cases are registered worldwide, particularly in developing countries, representing the second most common cause of mortality in women. Human Papillomaviruses (HPVs) are the primary etiologic agent of cervical carcinoma; HPV DNA can be found in more than 95% of these cancers (1). Since 1998, prevention and treatment strategy mainly rely on structured screening programs to detect and ablate pre-invasive disease. However, use of HPV testing is limited by social issues and currently the main obstacle is its high cost. Thus the development of vaccines that prevent HPV infection represent an important opportunity to prevent cervical cancer whilst a therapeutic immunization would be valuable in treating pre-malignant and malignant disease.
HPVs belong to a large family of small double-stranded DNA viruses that infect squamous epithelia. (For a recent comprehensive review on papillomaviruses see Howley, P M and Lowy, D R (2007) in: Fields Virology, fifth edition), eds.-in-chief Knipe, D. M. &. Howley, P. M. Lippincott Williams & Wilkins, Philadelphia Pa. 19106, USA, pp. 2299-2354 To date, more than 100 genotypes have been described, among which at least 35 types infect the genital tract. Although most of the HPV types produce benign lesions, a small subset of genotypes is strongly associated with the development of high-grade squamous intraepithelial lesions and cervical cancer. This subset has been identified as “high risk” and it is estimated that HPV-16 accounts for approximately 60% of cervical cancers, with HPV-18 adding another 10%-20%. Other high-risk types include types 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, and 73. Low-risk HPVs, such as HPV-6 and HPV-11, cause benign genital warts (90% begnin condylomata accuminata are HPV6 or 11 positive). Bivalent vaccines 16/18 eliminating the most common high-risk types may permit to overcome also the low-risk types. An ideal vaccine would protect against other HPV types through use of antigens from different types and/or antigens containing conserved regions.
The HPV genome encodes eight proteins. The late L1 and L2 genes code for capsid proteins; the early proteins E1 and E2 are responsible for viral replication and transcription, and E4 is involved in virus release from infected cells. The integration of high risk type HPV viral DNA into host genome results in a loss of E1 or E2 mediated transcriptional control and consequently in an over-expression of the E6 and E7 proteins responsible of the malignant transformation process (2). Structural protein L1 from high risk types represents an optimal target for prophylactic vaccines. On the other hand, E6 and E7 proteins are obvious therapeutic targets.
There are actually several prophylactic HPV vaccine formulations based upon the major viral capsid protein L1, either as a monomer, or as a virus like particle (VLP) from HPV 16 and 18 types and in some cases additional types (W094/00152, W094/20137, WO93/02184 and WO94/05792). VLPs may additionally comprise L2 proteins, for example, L2 based vaccines described in WO93/00436. These vaccines are highly immunogenic and appear safe; however their high cost does not permit generalized access to populations at risk and their HPV type specificity represents another limitation. Therefore, a remaining need exists to develop additional improved vaccines against HPV which should be inexpensive. Moreover vaccinations with antigen mainly induce an antibody specific response that is of little or no benefit on established HPV infection and related-disease.
The development of therapeutic vaccine relies not only on production of neutralising antibodies, but principally on the induction of specific cellular immune responses, that are key components for clearance of established infection. Thus, therapeutic vaccines are required to include some antigenic determinants derived from early HPV proteins rather than the late proteins. The early genes of the high-risk HPV types (E6 and E7) encode the main transforming proteins. These genes are capable of immortalization of epithelial cells and are thought to play a role in the initiation of the oncogenic process. The protein products of these early genes interfere with the normal function of tumour suppressor genes. HPV E6 is able to interact with p53, leading to its dysfunction, thereby impairing its ability to block the cell cycle when DNA errors occur. E6 also keeps the telomerase length above its critical point, protecting the cell from apoptosis. HPV E7 binds to retinoblastoma protein (pRb) and activates genes that start the cell cycle, leading to tissue proliferation. E6 and E7 proteins represent good targets and various approaches of HPV therapeutic vaccines have been described based on E6. In the last few years a number of peptide/protein-based or genetic immunization strategies have been described for the induction of HPV specific CTL activity. For a review of progress in the development of vaccines against HPV see ref (3). Attempts were made with DNA vaccines (plasmid DNA encoding HPV proteins) known to promote primarily a cellular response. Despite the fact that DNA vaccines work well in mouse models, numerous clinical trials have failed to provide proof of principle in man. Major drawbacks associated with a peptide-based approach include the problem of MHC-polymorphism and the risk of inducing T cell tolerance rather than T cell activation. Due to the induction of specific T cell tolerance, vaccination with a tumour-specific peptide has been shown to result in an enhanced outgrowth of the tumour. Immunization with larger proteins would overcome these problems, but this requires an efficient in vivo expression system and/or safe adjuvants for priming an efficient cellular immune response. Approaches involving recombinant viral vector vaccines are under development (Poxvirus, Adenovirus, Alphavirus, Poliovirus e Herpes Virus). Adenovirus based vaccine is described, for example, in US2007269409 (WO2004044176) which encodes the E6 or E7 protein of HPV. The adenovirus based vaccine is able to generate long term immunity; however, integration of HPV DNA into the host genome remains possible and may represent a safety limitation. In view of the above shortcomings the use of measles virus as a vector to express HPV antigens represents an original strategy to develop a prophylactic combined HPV-measles vaccine as well as a therapeutic HPV vaccine.
Immunisation Vectors Based on Measles Virus
Measles virus (MV) is a member of the family Paramyxoviridae. The non segmented genome of MV has an anti-message polarity which results in a genomic RNA which, when purified, is not translated either in vivo or in vitro and is not infectious. Transcription and replication of non-segmented (−) strand RNA viruses and their assembly as virus particles have been studied and reported (4). Transcription and replication of measles virus do not involve the nucleus of the infected cells but rather take place in the cytoplasm of said infected cells. The genome of the measles virus comprises genes encoding six major structural proteins from the six genes (designated N, P, M, F, H and L) and additional two-non structural proteins from the P gene. MV is a major cause of acute febrile illness in infants and young children. According to estimates of the World Health Organisation (WHO), one million young children die every year from measles. This high toll arises primarily in developing countries, but in recent years also industrialised countries such as the USA have been affected again by measles epidemics, primarily due to incomplete adherence to immunisation programs (5). At present, several live attenuated MV vaccine strains are in use (including the Schwarz, Moraten and Edmonston-Zagreb strains), almost all derived from the original Edmonston strain (6) by multiple passage in non human cells. MV vaccine is proven to be one of the safest, most stable, and effective human vaccines developed so far. Produced on a large scale in many countries and distributed at low cost through the Extended Program on Immunization (EPI) of WHO, this vaccine induces life-long immunity after a single injection (4, 7) and boosting is effective. Protection is mediated both by antibodies and by CD4 and CD8 T cells. Persistence of antibodies and CD8 cells has been shown for as long as 25 years after vaccination (7).
Martin Billeter and colleagues established an original and efficient reverse genetics procedure to generate non-segmented negative-strand RNA viruses from cloned deoxyribonucleic acid (cDNA) derived from Edmonston strain MV, as described in 8 and WO 97/06270, a scheme of the organization of the antigenomic p(+)MVEZ of measles virus is represented in FIG. 1. In the first place, these technologies allowed site directed mutagenesis enabling important insights in a variety of aspects of the biology of these viruses. Concomitantly, foreign coding sequences were inserted a) to allow localization of virus replication in vivo through marker gene expression, b) to develop candidate multivalent vaccines against measles and other pathogens, and c) to create candidate oncolytic viruses. The vector use of these viruses was experimentally encouraged by the pronounced genetic stability of the recombinants unexpected for RNA viruses, and by the high load of insertable genetic material, in excess of 6 kb. The known assets, such as the small genome size of the vector in comparison to DNA viruses, the extensive clinical experience of attenuated MV as vaccine with a proven record of high safety and efficacy, and the low production cost per vaccination dose are thus favourably complemented.
The recombinant measles virus nucleotide sequence must comprise a replicon having a total number of nucleotides which is a mutiple of six. The <<rule of six>> is expressed in the fact that the total number of nucleotides present in the recombinant cDNA finally amount to a total number of nucleotides which is a multiple of six, a rule which allows efficient replication of genome RNA of the measles virus.
The heterologous DNA is cloned in the MV vector within an Additional Transcription Unit (ATU) inserted in the cDNA corresponding to the antigenomic RNA of measles virus. The location of the ATU can vary along said cDNA: it is however located in such a site that it will benefit from the expression gradient of the measles virus. Therefore, the ATU or any insertion site suitable for cloning of the heterologous DNA sequence can be spread along the cDNA, with a preferred embodiment for an insertion site and especially in an ATU, present in the N-terminal portion of the sequence and especially within the region upstream from the L-gene of the measles virus and advantageously upstream from the M gene of said virus and more preferably upstream from the N gene of said virus.
The advantageous immunological properties of the recombinant measles viruses can be shown in an animal model which is chosen among animals susceptible to measles viruses, and wherein the humoral and/or cellular immune response against the heterologous antigen and/or against the measles virus is determined. Among such animals suitable to be used as model for the characterization of the immune response, the skilled person can especially use transgenic mice expressing CD46 specific receptor for MV, or in monkeys.
The technology permits to produce rescued viruses containing and stably expressing foreign genes suitable for use as combined MV vaccines. As a proof of concept, MV has been used to express antigens derived from SIV, HIV, hepatitis B, mumps, West Nile (WN) Virus and SARSCoV (9-12). In most of these studies, recombinant MVs that express heterologous antigens appeared to induce specific humoral neutralizing antibodies in a transgenic mouse model (13) and were shown to induce cellular immune responses to some proteins (9, 11). At the present, clinical trials with any recombinant vaccine candidate based on MV are only in the planning stage however experimental results support the hypothesis that MV combined vaccines should be as efficient in eliciting long-lasting immune protection against other pathogenic agents as against the vector virus itself. In fact, in the case of MV expressing WNV gpE, a complete protection up to six months has been documented in monkeys (14), and MV expressing a Dengue antigen induced long term production of neutralizing antibodies (15). Moreover, in transgenic mice and macaques, rescued recombinant MV was capable of inducing specific antibody responses to heterologous antigen in the presence of pre-existing immunity against MV (9, 11, 16).
Rescued live recombinant MV vaccines are easily produced on a large scale in most countries and can be distributed at low cost. Regarding safety, MV replicates exclusively in the cytoplasm, ruling out the possibility of integration into host DNA. These characteristics make rescued recombinant MV vaccine an attractive candidate to be used as a multivalent vaccination vector for HPV antigens. Adult populations, even already MV immunized individuals, may however also benefit from MV recombinant immunization because re-administering MV virus under the recombinant form of the present invention may result in a boost of anti-MV antibodies (11)
So far, no approach has been developed to produce a vaccine able to induce immunity against MV combined with immunity against HPV.
The invention relates in particular to the preparation of recombinant measles viruses, bearing heterologous nucleic acid encoding antigens from HPV.